Helper-dependent (HD) adenoviral vectors are devoid of all viral coding sequences and can efficiently transduce a wide variety of cell types from numerous species in a cell cycle independent manner. The first efficient and currently most widely used method of generating HD vector relies on coinfection of Cre-expressing 293 cells with the HD vector and a helper virus bearing a packaging signal flanked by IoxP sites. Cre-mediated packaging signal excision renders the helper viral genome unpackagable but still able to replicated and provide all functions in trans necessary for HD vector propagation. It has become clear that HD vectors can direct long-term transgene expression in vivo with negligible toxicity and appear to be ideally suited for many gene therapy applications. However, despite their tremendous potential, several obstacles currently hinder the progress of this technology. Specifically, the current systems for generating HD vectors are technically demanding, not easily scalable and not widely available. These obstacles severely limit meaningful efficacy and safety studies of these vectors in large animal models and their ultimate use in clinical applications. Therefore, the overall objective of this proposal is to develop improved systems as well as optimized standard operating procedures to efficiently and reliably generate large quantities of HD vectors. To accomplish this, we will investigate and optimize the factors that influence HD vector amplification efficiency, develop simple methods for large scale vector production and develop systems to further reduce helper virus contamination. We will also develop improved, certifiable reagents for generating vectors suitable for clinical applications, which will be made widely available. Successful completion of these objectives would permit application of this promising technology for the treatment of a wide variety of acquired and inherited diseases.